RNP complex formation : Prior to setting up the mixture for RNP complex formation, Cas9 was diluted to a working concentration of 50 pmol/µL and the sgRNAs pooled at concentrationsbetween 16.7 pmol/uL and 50 pmol/µL L, depending on the number of sgRNAsin the pool. The complete P3 nucleofector solution was created by mixing the NucleofectorSolution P3 with the provided Supplement at a 4.5:1 ratio. The Nucleofector solution, Cas9 and sgRNA pool were then mixed as outlined in the tables within the detailed protocol and incubated at room temperature for 15–20 .mins

For each 20 µL nucleofection, 2–4 x 105 cells were aliquot into a separate 1.5 mL microcentrifuge tube and 500 µL 1x PBS added to wash. Then centrifuged for 5 minutes at 350 x g, room temperature. The liquid was then aspirated as completely as possible without disrupting the cell pellet (We typically leave up to 2µL of wash solution without issue). The cell pellet was then resuspended in 20µL of RNP complexes. All of the cell-RNP solution was then transferred to one well of a 16-well Nucleocuvette strip for each sample and nucleofected in a Nucleofector 4D X unit using Program DS-150 following the manufactures instructions. The cells were recovered in each well of the 16-well strip by adding 100µl of CD34 culture media and transferring to a 96-well plate.